Bottom Line:
This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism.The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event.These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.

Affiliation: Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.

ABSTRACTThe small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide-sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPgammaS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPgammaS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles. These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.

Figure 5: Recombinant ARF1 restores GTPγS sensitivity to ARF-depleted cytosol. Unfractionated bovine brain cytosol (5 μl) and ARF-depleted cytosol (5-μl equivalents) were tested in the medial (A), trans (B), and TGN (C) assays in the presence of 2 μM GTPγS (solid bars) or in its absence (open bars). The indicated amounts of myr-rARF1 (5.7% myristoylated) or non–myr-rARF1 (hatched bar) were titrated into the assay. Data are a combination of two independent experiments. The maximum counts per minute for unfractionated cytosol were 7,764 ± 184 in the medial assay, 4,076 ± 64 and 2,960 ± 203 in the trans assay, 3,030 ± 204 and 3,389 ± 48 in the TGN assay. The maximum counts per minute for ARF-depleted cytosol were 4,866 ± 114 and 8,850 ± 15 in the medial assay, 2,184 ± 323 and 1,881 ± 15 in the trans assay, and 2,981 ± 153 and 2,498 ± 31 in the TGN assay. The acceptor membranes used in these experiments were different from the preparation used in Fig. 4 and exhibit less cytosol-independent inhibition of transport to the TGN by GTPγS (compare Figs. 4 C and 5 C).

Mentions:
To verify this, we examined the GTPγS sensitivity of each assay upon addition of purified recombinant myristoylated ARF1 (myr-rARF1). As shown in Fig. 5, inhibition is restored in all assays by addition of myr-rARF1 to ARF-depleted cytosol in the presence of GTPγS (black bars). Since only 5.7% of the myr-rARF1 is actually myristoylated (Kahn, R., personal communication), the concentrations of myr-rARF1 that cause inhibition are comparable to the concentration of endogenous ARFs in an assay containing 1–4 μl of CHO cytosol (1.6–6.4 ug/ml final at 40 ng ARF per μl of cytosol; reference 56). Addition of non– myr-rARF1 (hatched bar) had no effect, as expected, since myristoylation is required for the biological activities of ARF (6, 20). For a given amount of myr-rARF1, inhibition was consistently least in the medial assay and greatest in the TGN assay, correlating with the differential cytosol dependence of GTPγS inhibition (Fig. 1). These data confirm that the cytosolic GTPγS-sensitive inhibitory component in all three assays is ARF. ARF, therefore, is not directly required to support in vitro Golgi transport, but rather exerts a negative effect when constitutively activated by GTPγS.

Figure 5: Recombinant ARF1 restores GTPγS sensitivity to ARF-depleted cytosol. Unfractionated bovine brain cytosol (5 μl) and ARF-depleted cytosol (5-μl equivalents) were tested in the medial (A), trans (B), and TGN (C) assays in the presence of 2 μM GTPγS (solid bars) or in its absence (open bars). The indicated amounts of myr-rARF1 (5.7% myristoylated) or non–myr-rARF1 (hatched bar) were titrated into the assay. Data are a combination of two independent experiments. The maximum counts per minute for unfractionated cytosol were 7,764 ± 184 in the medial assay, 4,076 ± 64 and 2,960 ± 203 in the trans assay, 3,030 ± 204 and 3,389 ± 48 in the TGN assay. The maximum counts per minute for ARF-depleted cytosol were 4,866 ± 114 and 8,850 ± 15 in the medial assay, 2,184 ± 323 and 1,881 ± 15 in the trans assay, and 2,981 ± 153 and 2,498 ± 31 in the TGN assay. The acceptor membranes used in these experiments were different from the preparation used in Fig. 4 and exhibit less cytosol-independent inhibition of transport to the TGN by GTPγS (compare Figs. 4 C and 5 C).

Mentions:
To verify this, we examined the GTPγS sensitivity of each assay upon addition of purified recombinant myristoylated ARF1 (myr-rARF1). As shown in Fig. 5, inhibition is restored in all assays by addition of myr-rARF1 to ARF-depleted cytosol in the presence of GTPγS (black bars). Since only 5.7% of the myr-rARF1 is actually myristoylated (Kahn, R., personal communication), the concentrations of myr-rARF1 that cause inhibition are comparable to the concentration of endogenous ARFs in an assay containing 1–4 μl of CHO cytosol (1.6–6.4 ug/ml final at 40 ng ARF per μl of cytosol; reference 56). Addition of non– myr-rARF1 (hatched bar) had no effect, as expected, since myristoylation is required for the biological activities of ARF (6, 20). For a given amount of myr-rARF1, inhibition was consistently least in the medial assay and greatest in the TGN assay, correlating with the differential cytosol dependence of GTPγS inhibition (Fig. 1). These data confirm that the cytosolic GTPγS-sensitive inhibitory component in all three assays is ARF. ARF, therefore, is not directly required to support in vitro Golgi transport, but rather exerts a negative effect when constitutively activated by GTPγS.

Bottom Line:
This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism.The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event.These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.

Affiliation:
Department of Biochemistry and Molecular Biology, St. Louis University School of Medicine, St. Louis, Missouri 63104, USA.

ABSTRACTThe small GTPase ADP-ribosylation factor (ARF) is absolutely required for coatomer vesicle formation on Golgi membranes but not for anterograde transport to the medial-Golgi in a mammalian in vitro transport system. This might indicate that the in vivo mechanism of intra-Golgi transport is not faithfully reproduced in vitro, or that intra-Golgi transport occurs by a nonvesicular mechanism. As one approach to distinguishing between these possibilities, we have characterized two additional cell-free systems that reconstitute transport to the trans-Golgi (trans assay) and trans-Golgi network (TGN assay). Like in vitro transport to the medial-Golgi (medial assay), transport to the trans-Golgi and TGN requires cytosol, ATP, and N-ethylmaleimide-sensitive fusion protein (NSF). However, each assay has its own distinct characteristics of transport. The kinetics of transport to late compartments are slower, and less cytosol is needed for guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS) to inhibit transport, suggesting that each assay reconstitutes a distinct transport event. Depletion of ARF from cytosol abolishes vesicle formation and inhibition by GTPgammaS, but transport in all assays is otherwise unaffected. Purified recombinant myristoylated ARF1 restores inhibition by GTPgammaS, indicating that the GTP-sensitive component in all assays is ARF. We also show that asymmetry in donor and acceptor membrane properties in the medial assay is a unique feature of this assay that is unrelated to the production of vesicles. These findings demonstrate that characteristics specific to transport between different Golgi compartments are reconstituted in the cell-free system and that vesicle formation is not required for in vitro transport at any level of the stack.